Cannabis Micropropagation, Cost Analysis and Viruses
By Dr. Hope Jones, Chief Scientific Officer of C4 laboratories
This presentation will educate the global cannabis industry (including hemp) on tissue culture micropropagation by providing a summary that will show some of the essential concepts behind tissue culture. Virus and pathogen elimination and the importance of a good IPM regime.
As well as the economics of the micropropagation industry, design and implementation plans from the small growers to the large in need of understanding the true potential of tissue culture micropropagation, and how to implement and how to avoid costly mistakes.
California’s regulated adult use cannabis market has been up and running for around four months now and rumors of a potential supply bottleneck on the horizon are beginning to circulate. There are a number of factors that could have an impact on the cannabis supply in the market, most of which stem from changes in the distribution channels now that the state is implementing new regulations.
Those include a slow rollout in licensing cannabis businesses, new testing requirements, the supply carryover period prior to January 1stas well as new labeling and packaging regulations. In this piece, we are going to examine some of those rumors, see if there might be some truth to them and provide some guidance for what businesses can do to prepare for this.
A Slow Start to Licensing
This one is perhaps the most obvious factor to impact the supply chain in California. Much of the delays in licensing cannabis businesses came from the issue of local control, where businesses needed to get approval from their municipality before getting a state license. In the first month of the new market, it took Los Angeles weeks longer than other counties to begin licensing dispensaries. Whereas San Diego retailers saw a massive influx of customers right away, forcing them to buy up product to meet the high demand. Smaller producers also had trouble getting licenses as quickly as some of the larger ones.
Basically it all boils down to a slow start for the new market, according to Diane Czarkowski, co-founder of Canna Advisors. “The state is requiring businesses to get their local licenses before they can get their state license and that will create a delay in operators being able to bring products to market,” says Czarkowski. She says this is pretty typical of new markets, or when a market experiences dramatic changes quickly. “It could be a brand-new market, like in Hawaii, where the operators were ready with product, but there were no labs to test the products, which caused delays.” In addition to the licensing roll out being slow to start, the temporary licenses initially awarded to businesses are set to expire soon, by the end of April.
Stricter Rules to Come
The same logic goes for the testing regulations. New testing and labeling requirements, according to the Bureau of Cannabis Control regulating the market, will be phased in throughout 2018.
The state has already phased in cannabinoids, moisture content, residual solvent, pesticide, microbial impurities and homogeneity testing to some extent. On July 1st, the state will add additional residual solvent and pesticide testing as well as foreign material testing. At the end of 2018, they plan on requiring terpenoids, mycotoxins, heavy metals and water activity testing. All of those tests cost money and all of those tests could impact suppliers’ ability to bring product to market. “Oftentimes regulations require different types of testing to be done to products without recognizing that adequately completing those tests requires different methods, equipment, and standards,” says Czarkowski. “Most labs do not have all of the necessary components, and they are very costly. Producers could wait weeks to get test results back before they know if they can sell their products.”
Back when we spoke with Josh Drayton, deputy director of the California Cannabis Industry Association, about the upcoming changes to the California market, he voiced his concerns with the coming testing rules. “A lot of testing labs are concerned they are unable to test at the state’s threshold for some of these contaminants and pesticides; the detection limits seem very low,” says Drayton. “The testing portion will take years to work out, I am sure we will remove and add different pesticides and contaminants to the list.” California’s testing industry is, however, capable of adapting to changing rules, as they’ve done in the past on more than one occasion. It should be noted that many labs in the state are on the cutting edge of testing cannabis, working with The Bureau to implement the new rules.
Cannabis products made prior to December 31st, 2017, did not need to comply with the stricter testing rules that are coming in the next few months. This carryover period allowed dispensaries to have products on the shelves when the new market launched in the beginning of 2018. Retailers knew this rule meant they needed to stockpile product in the event of a supply bottleneck, and it appears much of that product is now sold and running out, according to Roy Bingham, founder and chief executive officer of BDS Analytics. “The true impact of licenses is starting to be felt since the carryover from December buying prior to the licensed market has been sold,” says Bingham. “Some of the major brands have consciously not applied for licenses. Some of that has to do with the flexibility the government has given them to wait.”
A fourth reason for a potential bottleneck could also come from packaging and labeling rules. “There will have to be many modifications to products to ensure they follow the new potency regulations, and many formulations will have to be modified in order to meet new regulations,” says Czarkowski. Distributor licenses, according to The Bureau, have a number of compliance documentation requirements, such as arranging for all product testing, quality assurance and packaging and label accuracy. Everything has to be packaged before it gets to a dispensary, which is a new rule California businesses need to comply with.
Pricing is the Indicator
There are a handful of reasons why prices could increase; some of them are more defined than others, the biggest factor being the tax burden passed on to consumers, where reports showed up to a 40% increase from last year. A price increase in the future could also come from The Bureau implementing testing regulations throughout 2018, as mentioned previously.
If prices were to surge enormously and very quickly, that might be an indicator that a shortage is fast approaching. A dramatic increase in price over this year could squeeze margins for smaller producers, forcing retailers to pass that burden on to consumers as well.“So yes, the rumors are true.”
According to Roy Bingham, there has been a significant increase in pricing in all categories at the retail level. “In January and February, we are seeing about 10% increases per month in average retail prices,” says Bingham. “If we look at concentrates in California during 2017, they averaged about $34 by the end of the year, whereas it was about $31 at the start of 2017. So in January, prices have increased up to $38, which is a bit above trend, but in fact we were seeing a trend upwards before January 1st as well.” Comparing that with edibles pricing, Bingham says we see a clear jump at the start of 2018. “It was basically flat in 2017, averaging $14 roughly almost straight-line across, dipped in December, then in January it jumped to $17 and then to $18 in February, a big increase and significantly more than concentrates,” says Bingham. He also says flower was hovering around $9 per gram in December 2017, but surged above $10 in February 2018.
According to Cannabis Benchmarks, the California wholesale averages surged in the summer of 2017 up to $1,631 by September, then reached their lowest point in December, with their spot index at $1,368. The Cannabis Benchmarks report underlines some important reasons for the changes in pricing, but they also attribute it to the new licensing system.
“Increasing operating expenses for businesses preparing to enter California’s licensed system in 2018 were key to propping up supply side rates in the first six months of 2017. New compliance requirements were being instituted to varying degrees by local governments, while market participants warily eyed draft regulations from state officials for guidance as to how to prepare their sites and facilities to meet under-construction regulatory mandates.”
Their report highlights some very important aspects of the supply chain. “Again, it is likely that the increased costs faced by operators up and down the supply chain exert some upward pressure on wholesale rates, preventing them from steep year-over-year declines that were observed in some of the other major Western markets,” reads the Cannabis Benchmarks report.
So How Can Businesses Prepare?
Well to start, producers should make sure their operations and product are clean and safe. Making sure your product will pass a pesticide test should be top of mind. Dispensaries should also be wise in selecting their suppliers, performing supplier quality audits or some form of verification that they meet your standards is key in a consistent supply chain.
Dr. Jon Vaught, chief executive officer of Front Range Biosciences, believes tissue culture could be a viable solution for some California producers. Using tissue culture, as a form of propagation instead of mothers and clones can be cleaner, cheaper and more efficient, thus allowing growers to keep up with demand and prevent a shortage.
Dr. Vaught says growers could look to tissue culture as a means to “mitigate risk to their supply chain and mitigate the risk of potential loss and improve their ability to efficiently grow their plant.” Maintaining a disease-free, sterile environment is a huge advantage in the cannabis market. “The real use of tissue culture is to provide disease free, clean, certified material, that has gone through a QA program,” says Dr. Vaught. “In greenhouses, the ability to control your environment is also critical because your margin of error is high. Variations in sunlight, weather, humidity all of these things have an impact in your plants. Technology can help monitor this.”
We’ve covered the basics of tissue culture previously on CIJ, with Dr. Hope Jones chief science officer of C4 Laboratories. She echoes many of Dr. Vaught’s points, firmly believing that, having existed for decades, tissue culture is an effective propagation tool for advanced breeders or growers looking to scale up.It is a complex supply chain that requires systems thinking.
It is important to note they don’t think growers should try this at home. Work with professionals, get the necessary funding, the training and facilities required if this is a project that interest you. “There’s a pretty big barrier to entry there,” Dr. Vaught urges. “The ability to manage thousands or millions of plants in a greenhouse increases risk, whereas in the lab, you’ve got a safe, secure, sterile environment, reducing risk of disease, making things easier to manage. The producers most successful at large scale are controlling those variables to the T.”
Ultimately, one segment of the market can’t prevent a bottleneck. It is a complex supply chain that requires systems thinking. Regulators need to work with producers, manufacturers, retailers, distributors, patients, consumers and laboratories to keep an eye on the overall supply chain flow.
Diane Czarkowski says the California market should prepare for this now if they haven’t already. “We have seen supply issues in every market going through a change. Other potential bottlenecks will occur because former distribution channels will be required to change,” says Czarkowski. “So yes, the rumors are true.”
In the first part of this series, we introduced some relevant terms and principles to tissue culture micropropagation and reviewed Dr. Hope Jones’ background in the science of it. In the second part, we went into the advantages and disadvantages of using mother plants to clone and why tissue culture could help growers scale up. In the third part of this series, we are going to examine the five steps that Dr. Jones lays out to successfully micropropagate cannabis plants from tissue cultures.
Cleaning – Stage 0
Micropropagation includes 5 stages. “Stage 0 is the preparation of mother plants and harvest of cuttings for the explant material,” says Dr. Jones. “To ensure the best chance of growing well in culture, those ladies [the mom’s] should be cleaned up and at their best. And hopefully not stressed by insects or pathogens.” She says growers should also make sure the plants are properly fertilized and watered before harvesting explants. “Obtaining the explants is done with a clean technique using new disposable blades and gloves,” says Dr. Jones. “Young shoot tips are harvested and placed in labeled, large Ziploc bags with a small amount of dilute bleach and surfactant solution, then placed in a cooler and taken to the lab.” This is a process that could be documented with record keeping and data logs to ensure the same care is taken for every explant. “Once in the lab, working in the sterile environment of the transfer hood, the cuttings are sterilized, typically with bleach and a little surfactant, and then rinsed several times with sterile water,” says Dr. Jones. Once they reach the sterile environment, Dr. Jones removes the leaves and cuts the stem down to individual nodes.
Establishment – Stage 1
Establishment essentially means waiting for the shoots to develop. Establishing the culture requires an absolutely sterile environment, which is why the first step is so important. “Proper explant disinfection is equally as important is the control parameters of the facility itself,” says Dr. Jones. Mother plants are not grown in sterile facilities, but in an environment that is invariably contaminated with dust, which harbors micro-organisms, insects and other potential sources of contamination, including human handling. We discussed some of this in Part 2.
Explants, once sterilized and placed in the culture vessel, must establish to the new aseptic conditions. “Basically Stage 0 ends when the explants are cleaned and placed in the vessel. Stage 1 begins on the shelf while we patiently sit, watch and wait for the shoot growth,” says Dr. Jones. “Successful establishment means we properly disinfected the explants because the cultures do not become contaminated with bacteria or fungi and new shoot growth emerges.”
Multiplication – Stage 2
This stage is rather self-explanatory as multiplication simplified means generating many more shoots per explant. In order to create a large number of plants needed for meeting the demand of weekly clone orders, Dr. Jones can break up, or subculture, one explant that contains multiple numerous new shoots. “Let’s say one vessel, which originally started with 4 explants each developed four new shoots. Working in the hood, I remove each explant from the vessel and place it on a sterile petri dish. Now I can divide each explant into 4 new explants and then place the four new explant cuttings into their own vessel. In this example, we started with one vessel with 4 explants,” says Dr. Jones. “Which when subcultured 4-6 weeks later, we now have 4 vessels with 16 plants.” This is instrumental in understanding how tissue culture micropropagation can help growers scale without the need for a ton of space and maintenance. From a single explant, you can potentially generate 70,000 plants after 48 weeks, according to Dr. Jones. “Starting with not 1, but 10 or 20 explants would significantly speed up multiplication.” Using tissue culture effectively, one can see how a grower can exponentially increase their production.
Rooting – Stage 3
“When the decision is made to move cultures to the rooting stage, we typically need to subculture the plantlets to a different media formulated to induce rooting,” says Dr. Jones. “In some instances, the media is very dark, and that’s because of the addition of activated charcoal.” Using activated charcoal, according to Dr. Jones, helps darken the rooting environment, which closely mimics a normal rooting environment. “It helps remove high levels of cytokinin and other possible inhibitory compounds,” says Dr. Jones. Cytokinins are a type of plant growth hormone commonly used to promote healthy shoot growth, but it is important to make sure the culture contains the right ratio of hormones, including cytokinin and auxin for maximum root and shoot development. Dr. Jones suggests that growers research their own media formulation to ensure nice, healthy roots develop and that no tissue dies in the process. “With everything I grow in culture, when it comes to media, in any stage and with all new strains, I run some simple experiments in order to refine the media used,” says Dr. Jones. She puts a special focus on the concentrations and ratios of plant hormones in formulating her medias.
“We commonly think of auxin’s role in rooting, but it’s also important in leaves and acts as a regulator of apical shoot dominance,” says Dr. Jones. “So having no auxin may not be ideal for the shooting media used in Stages 1 and 2.” Auxin is a plant hormone that can help promote the elongation of cells, an important step in any plant’s growth. “And cytokinins are typically synthesized in the root and moves through xylem to shoots to regulate mitosis as well as inducing lateral bud branching, so again finding that nice balance between these two hormones is key.”
Acclimation & Hardening Off – Stage 4
“When plants have developed good looking healthy roots, it’s time to pop the top,” says Dr. Jones. This means opening the vessel, another risk for contamination, which is why having a clean environment is so crucial. “The location of these vessels needs to be tightly controlled for light, relative humidity, temperature and cleanliness.” In the culture, sugar is a main ingredient in the medium, because the growing explants are not very photosynthetically active. “By opening the lid of the vessel, carbon dioxide is introduced to the environment, which promotes and enhances photosynthesis, really getting the plants ready for cultivation.”
The very final step in tissue culture micropropagation is hardening, which involves the formation of the waxy cuticle on the leaves of the plant, according to Dr. Jones. This is what preps the plant to actually survive in an unsterile environment. “The rooted plants are removed from the culture vessel, the media washed off and placed in a potting mix/matrix or plug and kept in high humidity and low light,” says Dr. Jones. “Now that there is no sugar, contamination is no longer a threat, and these plants can be moved to the grow facility.” She says conditioning these plants can take one or two weeks. Over that time, growers should gradually increase light intensity and bring down the relative humidity to normal growing conditions.
Overall, this process, if done efficiently, can take roughly eleven weeks from prepping the explants to acclimation and hardening. If growers perform all the steps correctly and with extra care to reduce risks of contamination, one can produce thousands of plants in a matter of weeks.
In the fourth and final part of this series, we are going to dive into implementation. In that piece, we will discuss design principles for tissue culture facilities, equipment and instrumentation and some real-world case studies of tissue culture micropropagation.
In the first part of this series, we introduced Dr. Hope Jones, who took her experience in tissue culture from NASA and brought it to the cannabis industry and C4 Laboratories. We discussed some of the essential concepts behind tissue culture and defined a few basic terms like micropropagation, totipotency, explants and cloning. Now let’s get into some of the issues with cloning from mother plants and the advantages that come with using tissue culture for propagating and cultivating cannabis.
Time & Resources
Taking cuttings from mother plants is arguably the most popular method of propagating cannabis plants. It is a process that requires significant real estate, resources and labor. “Moms can take up a great deal of space that is not contributing directly to production,” says Dr. Jones. “I know from experience that scaling up production and/or adding new strains to the production line requires significant time and resources to raise and maintain new healthy and productive mother plants.” Each mother plant produces a limited number of clones per harvest period and over the course of her life cycle.
By using tissue culture, a cultivator can generate an almost infinite number of clones from one plant cutting. With so many growers calculating their costs-per-square-foot, micropropagation is an effective tool to save space, labor and time, thus increasing profit margins. “Just to put it in perspective: Holly Scoggins’ book Plants From Test Tubes, cites a Day Lily cultivator who uses micropropagation to produce 1,000 plants in 30 square feet of shelf space each week,” says Dr. Jones. “Using conventional methods, one would need a half-acre to produce the same amount of plants.” Cultivators can produce a much greater number of plants-per-square-foot by using micropropagation effectively.
Early Health & Vigor
Most tissue culture methods use sterilized vessels that contain sugar-rich media to support growth of plantlets before they can photosynthesize on their own. “The media is prepped, poured into vessels, and placed in an autoclave (or pressure cooker) where it is subjected to high temps and pressure to achieve proper sterility.”
The sterile environment and rich growth media supplies plantlets with an abundance of everything they need. “When plantlets emerge from culture, they are pathogen-free, with a stockpile of food and nutrient reserves that support rapid growth and vigor, superior to conventional cuttings,” says Dr. Jones.
Stress & Disease
As any grower knows, mother plants can sometimes experience stress and disease. This might come in the form under or over-watering, heat stress, spider mites, whiteflies, mold and viruses. “Any stress or infection that a mother plant is subjected too can impact progeny health and productivity in a couple of ways,” says Dr. Jones.
For example, diseases like powdery mildew and tobacco mosaic virus are often systemic, meaning that pathogens have spread to almost every tissue in the plant. Once infected, it is impossible to completely eliminate pathogens from tissues. Therefore any cuttings made from a diseased mother plant, even if they look perfectly healthy, will also be infected and can eventually present disease symptoms like reduced productivity and/or plant death, according to Dr. Jones.
How does tissue culture get around this problem? Remember that explants (small tissue samples used as starting material) can be extracted from any part of the plant. Meristematic cells in shoot tips and leaves are the source of new plant growth. Dr. Jones explains that these cells, and the first set of primordial leaves are not connected directly to the vascular tissue, the plant’s transport system by which pathogens spread. Therefore, meristematic cells tend to be disease-free, whatever the condition of the mother. It takes a sharp blade, a dissecting microscope, and a lot of experience to learn, but as Dr. Jones explains, “harvesting explants from meristems is a routine micropropagation technique used by ‘Big Horticulture.’ One example is the strawberry. Viruses and pathogens are so prevalent that the strawberry industry must use meristematic culture to ensure pathogen free progeny.”
Now let’s talk about epigenetics. We know that plants don’t have the option of physically moving away from stress or predation. Instead, they have evolved sophisticated ways of changing their own biology to adapt to and/or protect themselves. “Consider what happens to a mom exposed to a pathogen. The infected plant will start expressing (turning on) genes and making proteins that contribute to pathogen resistance,” says Dr. Jones. “These changes to gene expression are partly regulated by epigenetic modifications, chemical changes to DNA that increase or decrease the likelihood a cell will express a particular gene, but that do not actually modify the gene itself. Like annotations to a piece of music, epigenetic modifications don’t change the notes but rather how loud or soft, quickly or slowly the notes are played.”
This is where it gets interesting. “Epigenetic modifications can be systemic and long lived. Plants infected by a pathogen or stressed by drought will present widespread epigenetic modifications to their DNA,” says Dr. Jones. “For an annual plant like cannabis, those modifications are relatively permanent. Thus a cutting from a mom having drought or pathogen adapted epigenetic programming will inherit that modified DNA and behave as if it were experiencing that stress, whether present or not.”
In the wild, this adaptability is critical for plant survival and reproduction, but to a grower, this is a less-than-ideal scenario. “The epigenetic modifications allowed the mother to tolerate the stress, which is great from the perspective of survival and fitness, but it comes at a cost. Some of the finite energy and resources that usually support plant growth and reproduction are instead channeled to stress response,” says Dr. Jones. This trade off results in reduction in overall plant yield and quality. “Those epigenetic changes result in a new phenotype for that mother,” says Dr. Jones. “All cuttings from her will reflect the new phenotype. This is one major mechanism underlying what many in the cannabis industry (incorrectly) call ‘genetic drift,’ or the loss of vigor over successive clonal generations.”
This is again where tissue culture can be such a game changer. The process of dedifferentiation, as explained in part 1 of this series, can rejuvenate a “tired” mother plant by inducing a kind of reboot– clearing accumulated epigenetic modifications that negatively impact progeny vigor and productivity. In the third part of this series, we will discuss the five stages of micropropagation, detailing the process of how you can grow plantlets in tissue culture. Stay tuned for more!
Dr. Hope Jones, chief scientific officer of C4 Laboratories, believes there are a number of opportunities for cannabis growers to scale their cultivation up with micropropagation. In her presentation at the CannaGrow conference recently, Dr. Jones discussed the applications and advantages of tissue culture techniques in cannabis growing.
Dr. Jones’ work in large-scale plant production led her to the University of Arizona Controlled Environment Agriculture Center (CEAC) where she worked to propagate a particularly difficult plant to grow- a native orchid species- using tissue culture techniques. With that experience in tissue culture, hydroponics and controlled environments, she took a position at the Kennedy Space Center working for NASA where she developed technologies and protocols to grow crops for space missions. “I started with strawberry TC [tissue culture], because of the shelf life & weight compared with potted plants, plus you can’t really ‘water’ plants in space- at least not in the traditional way,” says Dr. Jones. “Strawberries pack a lot of antioxidants. Foods high in antioxidants, I argued, could boost internal protection of astronauts from high levels of cosmic radiation that they are exposed to in space.” That research led to a focus on cancer biology and a Ph.D. in molecular & cellular biology and plant sciences, culminating in her introduction to the cannabis industry and now with C4 Labs in Arizona.
Working with tissue culture since 2003, Dr. Jones is familiar with this technology that is fairly new to cannabis, but has been around for decades now and is widely used in the horticulture industry today. For example, Phytelligence is an agricultural biotechnology company using genetic analysis and tissue culture to help food crop growers increase speed to harvest, screen for diseases, store genetic material and secure intellectual property. “Big horticulture does this very well,” says Dr. Jones. “There are many companies generating millions of clones per year.” The Department of Plant Sciences Pomology Program at the Davis campus of the University of California uses tissue culture with the Foundation Plant Services (FPS) to eliminate viruses and pathogens, while breeding unique cultivars of strawberries.
First, let’s define some terms. Tissue culture is a propagation tool where the cultivator would grow tissue or cells outside of the plant itself, commonly referred to as micropropagation. “Micropropagation produces new plants via the cloning of plant tissue samples on a very small scale, and I mean very small,” says Dr. Jones. “While the tissue used in micropropagation is small, the scale of production can be huge.” Micropropagation allows a cultivator to grow a clone from just a leaf, bud, root segment or even just a few cells collected from a mother plant, according to Dr. Jones.
The science behind growing plants from just a few cells relies on a characteristic of plant cells called totipotency. “Totipotency refers to a cell’s ability to divide and differentiate, eventually regenerating a whole new organism,” says Dr. Jones. “Plant cells are unique in that fully differentiated, specialized cells can be induced to dedifferentiate, reverting back to a ‘stem cell’-like state, capable of developing into any cell type.”
Cannabis growers already utilize the properties of totipotency in cloning, according to Dr. Jones. “When cloning from a mother plant, stem cuttings are taken from the mother, dipped into rooting hormone and two to five days later healthy roots show up,” says Dr. Jones. “That stem tissue dedifferentiates and specializes into new root cells. In this case, we humans helped the process of totipotency and dedifferentiation along using a rooting hormone to ‘steer’ the type of growth needed.” Dr. Jones is helping cannabis growers use tissue culture as a new way to generate clones, instead of or in addition to using mother plants.
With cannabis micropropagation, the same principles still apply, just on a much smaller scale and with greater precision. “In this case, very small tissue samples (called explants) are sterilized and placed into specialized media vessels containing food, nutrients, and hormones,” says Dr. Jones. “Just like with cuttings, the hormones in the TC media induce specific types of growth over time, helping to steer explant growth to form all the organs necessary to regenerate a whole new plant.”
Having existed for decades, but still so new to cannabis, tissue culture is an effective propagation tool for advanced breeders or growers looking to scale up. In the next part of this series, we will discuss some of issues with mother plants and advantages of tissue culture to consider. In Part 2 we will delve into topics like sterility, genetic reboot, viral infection and pathogen protection.
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